Mutagenicity of bitumen and asphalt fumes.

The mutagenicity of asphalt fumes was tested with the Salmonella bioassays. The aim was to investigate if recycled additives modify the genotoxicity of emissions. Recycling of old asphalt is increasing, and we studied also the mutagenicity of emissions sampled during the re-use of asphalt. The composition of vapours and fumes were analysed by gas chromatography and by liquid chromatography. Bitumens containing coal fly ash (CFA) or waste plastics were heated to the paving temperatures in the laboratory. In the field, bitumen fumes were collected during paving of stone mastic asphalts (lime or CFA as a filler), remixing of stone mastic asphalt (lime or CFA as a filler), and of asphalt concrete. All the lab-generated vapour fractions were non-mutagenic. The particulate fractions were mutagenic with TA98 in the presence of the S9 activation. In addition, the lab-fumes from bitumen containing waste plastics were positive with both strains without S9. Only particulate fractions sampled in the field were tested. They were mutagenic with and without metabolic activation with both strains. The mutagenic potency of the field samples was higher than that of the lab-generated fumes without S9, and the remixing fumes were more mutagenic than the normal paving and lab-generated fumes with S9. The use of inorganic additive, CFA, did not change the mutagenicity of the fumes, whereas the organic additive, waste plastics, increased the mutagenicity of the laboratory emissions significantly.

Expression and prognostic significance of catalase in malignant mesothelioma.

BACKGROUND: Free radicals and antioxidant enzymes (AOEs) may play a critical role in cell proliferation and in the resistance of malignant cells against cytotoxic drugs and radiation. Malignant mesothelioma is a resistant tumor with high levels of manganese superoxide dismutase, a central superoxide scavenging AOE. In the current study, the authors assessed the expression and prognostic role of catalase, an important hydrogen peroxide scavenging AOE, in malignant pleural mesothelioma. METHODS: Catalase expression was investigated by immunohistochemistry in 5 cases of nonmalignant healthy pleura and in tumor tissue of 32 mesothelioma patients, and by Western blot in 7 continuous human mesothelioma cell lines. The distribution of catalase in mesothelioma cells was assessed by immunoelectron microscopy. Furthermore, to investigate the effect of catalase inhibition in the drug resistance of these cells in vitro, the authors exposed mesothelioma cells with the highest catalase level to epirubicin with and without aminotriazole pretreatment. RESULTS: Nonmalignant mesothelial cells showed no catalase immunoreactivity whereas most mesothelioma cases (24 of 32, 75%) were catalase positive, 17 cases (53%) showing moderate or high expression. Higher catalase expression in mesothelioma was associated with a better prognosis, mean survival rate from diagnosis being 6 and 24 months for negative/low expression and moderate/high expression, respectively. Furthermore, a coordinately high expression of both manganese-superoxide dismutase (Mn-SOD) and catalase predicted even more favorable outcome of the mesothelioma patients. Catalase also could be detected in all mesothelioma cell lines, the most resistant cell line showing the highest protein expression and compartmentalization of catalase mainly to peroxisomes. Aminotriazole inhibition of catalase had a marginal effect on the toxicity caused by epirubicin. CONCLUSIONS: Catalase may have multifactorial effects in malignant cells; high catalase and/or coordinated high expression of Mn-SOD and catalase may decrease tumor progression by modulating the cellular redox state, but enhanced antioxidant capacity of mesothelioma cells also may protect tumor cells against exogenous oxidants, at least in vitro.

Gene expression profiling of malignant mesothelioma cell lines: cDNA array study.

To reveal genes relevant for malignant mesothelioma (MM), we carried out cDNA array experiments on 4 MM cell lines and 2 primary mesothelial cell cultures established from pleural fluid of non-cancer patients. Human cancer gene filters including 588 genes were used for the cDNA array experiments. Our study revealed 26 over-expressed genes that play a role in the regulation of cell fate, cell cycle, cell growth and DNA damage repair and 13 under-expressed genes encoding growth factors, receptors and proteins involved in cell adhesion, motility and invasion to be common to 3 or 4 MM cell lines. We confirmed the cDNA array results using RT-PCR for 5 of the over-expressed genes and for 3 of the under-expressed genes. Our study presents gene expression profiles in MM cell lines and shows the involvement of several genes, such as those encoding JAGGED1, ser/thr protein kinase NIK, Ku80 and cyclin D2, novel in MM.

Modulation of cell and DNA damage by poly(ADP)ribose polymerase in lung cells exposed to H(2)O(2) or asbestos fibres.

Poly(ADP)ribose polymerase (PARP) may participate in cell survival, apoptosis and development of DNA damage. We investigated the role of PARP in transformed human pleural mesothelial (MeT-5A) and alveolar epithelial (A549) cells exposed from 0.05 to 5mM hydrogen peroxide (H(2)O(2)) or crocidolite asbestos fibres (1-10 microg/cm(2)) in the presence and absence of 3-aminobenzamide (ABA), a PARP inhibitor. The cells were investigated for the development of cell injury, DNA single strand breaks and depletion of the cellular high-energy nucleotides. Compared to H(2)O(2), fibres caused a minor decrease in cell viability and effect on the cellular high-energy nucleotide depletion, and a marginal effect on the development of DNA strand breaks when assessed by the single cell gel electrophoresis (the Comet assay). Inhibition of PARP transiently protected the cells against acute H(2)O(2) related irreversible cell injury when assessed by microculture tetrazolium dye (XTT) assay and potentiated oxidant related DNA damage when assessed by the Comet assay. However, PARP inhibition had no significant effect on fibre-induced cell or DNA toxicity with the exception of one fibre concentration (2 microg/cm(2)) in MeT-5A cells. Apoptosis is often associated with PARP cleavage and caspase activation. Fibres did not cause PARP cleavage or activation of caspase 3 further confirming previous results about relatively low apoptotic potential of asbestos fibres. In conclusion, maintenance of cellular high-energy nucleotide pool and high viability of asbestos exposed cells may contribute to the survival and malignant conversion of lung cells exposed to the fibres.

Proliferation, apoptosis, and manganese superoxide dismutase in malignant mesothelioma.

Proliferation and apoptotic indices of tumour cells may have important prognostic significance. Manganese superoxide dismutase (MnSOD), an important anti-oxidant enzyme, has been shown to decrease proliferation of malignant cells transfected with the MnSOD gene. The aim of the present study was to investigate the indices of cell proliferation and apoptosis and their prognostic significance in human mesothelioma and to assess the effect of MnSOD on the proliferation and apoptosis of the mesothelioma cells expressing high constitutive MnSOD activity. Tissue sections from 35 subjects with malignant pleural mesothelioma were studied for cell proliferation by Ki-67 immunohistochemistry and for apoptosis by the TUNEL assay. In additional experiments, 2 mesothelioma cell lines expressing either low (M14K) or high (M38K) MnSOD levels were assessed for proliferative and apoptotic responses to epirubicin. The median proliferation and apoptotic indices of the mesothelioma tissue were 8.2% and 0.75%, respectively. Patients with a high proliferation (>8%) or apoptotic index (>0.75%) showed a worse prognosis (p < 0.001). MnSOD expression was inversely correlated with cell proliferation (p = 0.02). Our cell line experiments indicated that cells expressing high MnSOD levels were more resistant to apoptosis and showed lower proliferation when exposed to epirubicin in vitro. These findings show that high proliferation and apoptosis are associated with a poor prognosis of mesothelioma and that a high MnSOD level is associated with low proliferation of tumour cells. Furthermore, experiments with cultured mesothelioma cells suggest the importance of MnSOD in the proliferation and apoptosis caused by drug exposure.

Docetaxel and irinotecan (CPT-11) in the treatment of malignant pleural mesothelioma--a feasibility study.

We chose to treat malignant pleural mesothelioma with a combination of docetaxel and irinotecan (CPT-11), because there have been preliminary reports that CPT-11 is active against mesothelioma, and docetaxel and CPT-11 were the most active agents in our in vitro experiments in human mesothelioma cell lines. Fifteen previously untreated patients with pleural mesothelioma (IMIG Stage III-IV) were given docetaxel 60 mg/m2 followed by CPT-11 190 mg/m2 on day 1, repeated every 3 weeks. All the patients were evaluable for toxicity and 13 patients were evaluated for response. No objective responses (complete or partial) were achieved, but there were two minor responses (overall response rate 15%) each of a duration of 4 months. Three patients had stable disease (23%); median time to progression was 7 months. Median survival in all the patients was 8.5 months from the first chemotherapy cycle and 11 months from diagnosis. Toxicity was severe with seven of 15 patients suffering neutropenic fever and six of 15 patients grade 3-4 diarrhea. The trial was discontinued because of toxicity and lack of activity. We do not recommend the combination of docetaxel and CPT-11 using the schedule presented here for further investigation in malignant mesothelioma. However, CPT-11 and docetaxel, individually, still warrant further study in this disease, especially in combination with cisplatin.

In vitro sensitivity of normal human mesothelial and malignant mesothelioma cell lines to four new chemotherapeutic agents.

In this study, we used four human mesothelioma cell lines (M14K, M24K, M25K and M38K), one transformed human mesothelial cell line (MeT-5A) and one primary mesothelial culture (UPL) to test for in vitro sensitivity to docetaxel, paclitaxel, SN-38 [an active metabolite of irinotecan (CPT-11)] and gemcitabine, as single agents. Subconfluent cell cultures were treated with 2x10(-9), 5x10(-9), 10(-8), 2x10(-8) and 5x10(-8) M concentrations of each drug for 48 h. The sensitivity was measured in terms of cell viability using the Trypan blue exclusion method. All four drugs were potent inhibitors of mesothelioma cell growth, but cell lines from different patients diverged in their sensitivity to the individual agents. In most cases docetaxel, paclitaxel and SN-38 were more potent killers of mesothelioma cells than gemcitabine. The induction of DNA damage was investigated using the Comet assay; cells from two cell lines (M14K and M25K) were treated with subtoxic 10(-8) M concentrations of each drug for 4, 24 and 48 h. Each of the agents caused a slight increase in DNA single-strand breaks at a concentration of 10(-8) M.

Antioxidant defense mechanisms of human mesothelioma and lung adenocarcinoma cells.

The development of drug resistance of tumors is multifactorial and still poorly understood. Some cytotoxic drugs generate free radicals, and, therefore, antioxidant enzymes may contribute to drug resistance. We investigated the levels of manganese superoxide dismutase (Mn SOD), its inducibility, and its protective role against tumor necrosis factor-alpha and cytotoxic drugs (cisplatin, epirubicin, methotrexate, and vindesin) in human pleural mesothelioma (M14K) and pulmonary adenocarcinoma (A549) cells. We also studied other major antioxidant mechanisms in relation to oxidant and drug resistance of these cells. A549 cells were more resistant than M14K cells toward both oxidants (hydrogen peroxide and menadione) and all the cytotoxic drugs tested. M14K cells contained higher basal Mn SOD activity than A549 cells (28.3 +/- 3.4 vs. 1.8 +/- 0.3 U/mg protein), and Mn SOD activity was significantly induced by tumor necrosis factor-alpha only in A549 cells (+524%), but the induction did not offer any protection during subsequent oxidant or drug exposure. Mn SOD was not induced significantly in either of these cell lines by any of the cytotoxic drugs (0.007-2 microM, 48 h) tested when assessed by Northern blotting, Western blotting, or specific activity. A549 cells contained higher catalase activity than M14K cells (7.6 +/- 1.3 vs. 3.6 +/- 0.5 nmol O(2). min(-1). mg protein(-1)). They also contained twofold higher levels of glutathione and higher immunoreactivity of the heavy subunit of gamma-glutamylcysteine synthetase than M14K cells. Experiments with inhibitors of gamma-glutamylcysteine synthetase and catalase supported our conclusion that mechanisms associated with glutathione contribute to the drug resistance of these cells.

Apoptosis and expression of apoptosis regulating proteins bcl-2, mcl-1, bcl-X, and bax in malignant mesothelioma.

We investigated apoptosis and the expression of bcl-2, mcl-1, bcl-X, and bax in histological sections from 35 malignant mesotheliomas and 21 metastatic adenocarcinomas. Moreover, the expression of bcl-2, mcl-1, bcl-X, and bax were assessed by Western blotting in nonmalignant human mesothelial cells (Met5A) and seven malignant cell lines. The apoptotic index in mesotheliomas was 1.07+/-1.14%. Patients with mesotheliomas showing a high apoptotic index (> or =0.75%) had a worse prognosis (P = 0.008). bcl-2 positivity was observed in only seven cases, but bcl-X, mcl-1, and bax positivity was seen in all of them. In immunoblotting experiments, all mesothelioma cell lines were negative for bcl-2 but positive for bcl-X, mcl-1, and bax. The apoptotic index in bcl-2-negative mesotheliomas was 1.25+/-1.24% and in bcl-2-positive ones, 0.47+/-0.42% (P = 0.014). The apoptotic index did not significantly associate with bcl-X, mcl-1, or bax expression (P = 0.19, P = 0.25, and P = 0.46, respectively). No significant difference was observed in apoptosis or expression of bcl-2, bcl-X, or bax between malignant mesotheliomas and metastatic adenocarcinomas. The former, however, showed more often weak mcl-1 immunoreactivity (P = 0.01). The results show that the extent of apoptosis may influence patient prognosis. bcl-2 is inversely associated with the apoptotic index but is relatively infrequently expressed in malignant mesotheliomas. Widespread expression of bcl-X, mcl-1, and bax suggests that these proteins may also take part in apoptosis regulation in mesotheliomas.

Simian virus 40 (SV40)-like DNA sequences not detectable in finnish mesothelioma patients not exposed to SV40-contaminated polio vaccines.

Occupational asbestos exposure can be demonstrated in 80% of mesothelioma cases. A possible role of simian virus 40 (SV40) in the etiology of mesothelioma was raised because several studies reported the presence and expression of SV40-like DNA sequences in human mesotheliomas. It is also known that expression of SV40 large T antigen inhibits cellular Rb and p53. This suggests that SV40 might render infected cells more susceptible to asbestos carcinogenicity. The SV40-like sequences are suggested to have arisen from contaminated polio vaccines. Millions of people in the United States and most European countries were inoculated with SV40-contaminated polio vaccine in 1955-1963. However, in Finland, where polio vaccination started in 1957, no SV40-contaminated vaccine was used. We used a polymerase chain reaction-based method to test for the presence of SV40-like sequences in DNA extracted from the frozen tumor tissues of 49 Finnish mesothelioma patients, most of whom had been occupationally exposed to asbestos. All of the Finnish tumor tissues tested negative for SV40-like sequences. The results suggest that the SV40-like sequences detected in mesothelioma tissue in some previous studies may indeed originate from SV40-contaminated polio vaccines. It is a matter of speculation whether the absence of SV40 infection has contributed to the relatively low incidence of mesothelioma in Finland (1/10(5) in 1990-1995).

Generation of reactive oxygen species by human mesothelioma cells.

Malignant mesothelioma cells contain elevated levels of manganese superoxide dismutase (MnSOD) and are highly resistant to oxidants compared to non-malignant mesothelial cells. Since the level of cellular free radicals may be important for cell survival, we hypothesized that the increase of MnSOD in the mitochondria of mesothelioma cells may alter the free radical levels of these organelles. First, MnSOD activity was compared to the activities of two constitutive mitochondrial enzymes; MnSOD activity was 20 times higher in the mesothelioma cells than in the mesothelial cells, whereas the activities of citrate synthase and cytochrome c oxidase did not differ significantly in the two cell lines. This indicates that the activity of MnSOD per mitochondrion was increased in the mesothelioma cells. Superoxide production was assayed in the isolated mitochondria of these cells using lucigenin chemiluminescence. Mitochondrial superoxide levels were significantly lower (72%) in the mesothelioma cells compared to the mesothelial cells. Oxidant production in intact cells, assayed by fluorimetry using 2',7'-dichlorodihydrofluorescein as a fluorescent probe, did not differ significantly between these cells. We conclude that mitochondrial superoxide levels are lower in mesothelioma cells compared to nonmalignant mesothelial cells, and that this difference may be explained by higher MnSOD activity in the mitochondria of these cells. Oxidant production was not different in these cells, which may be due to the previously observed increase in H2O2-scavenging mechanisms of mesothelioma cells.

Asbestos induction of extended lifespan in normal human mesothelial cells: interindividual susceptibility and SV40 T antigen.

Normal human mesothelial cells from individual donors were studied for susceptibility to asbestos-induction of apoptosis and generation of an extended lifespan population. Such populations were generated after death of the majority of cells and arose from a subset of mesothelial cultures (4/16) whereas fibroblastic cells (5/5) did not develop extended lifespan populations after asbestos exposure. All mesothelial cultures were examined for the presence of SV40 T antigen to obtain information on (i) the presence of SV40 T antigen expression in normal human mesothelial cells and (ii) the relationship between generation of an extended lifespan population and expression of SV40 T antigen. Immunostaining for SV40 T antigen was positive in 2/38 normal human mesothelial cultures. These cultures also had elevated p53 expression. However, the two isolates expressing SV40 T antigen did not exhibit enhanced proliferative potential or develop an extended lifespan population. Asbestos-generated extended lifespan populations were specifically resistant to asbestos-mediated but not to alpha-Fas-induced apoptosis. Deletion of p16Ink4a was shown in 70% of tumor samples. All mesothelioma cell lines examined showed homozygous deletion of this locus which extended to exon 1beta. Extended lifespan cultures were examined for expression of p16Ink4a to establish whether deletion was an early response to asbestos exposure. During their rapid growth phase, extended lifespan cultures showed decreased expression of p16Ink4a relative to untreated cultures, but methylation was not observed, and p16Ink4a expression became elevated when cells entered culture crisis. These data extend the earlier observation that asbestos can generate extended lifespan populations, providing data on frequency and cell type specificity. In addition, this report shows that generation of such populations does not require expression of SV40 T antigen. Extended lifespan cells could represent a population expressing early changes critical for mesothelioma development. Further study of these populations could identify such changes.

DNA single strand breaks induced by asbestos fibers in human pleural mesothelial cells in vitro.

The mechanisms of the cellular effects and DNA damage caused by asbestos fibers in human mesothelial cells are not well understood. We exposed transformed human pleural mesothelial cells to 1-4 microg/cm2 crocidolite and to 10-100 ng/ml tumor necrosis factor alpha for up to 48 hr and studied the induction of DNA damage using the Comet assay. As a positive control, 100 microM H2O2 was used. The DNA single strand breaks were assessed as the mean tail moments and as distributions of the tail DNA in the cell. The Comet assay showed significant but reversible increases in the mean tail moments, but not in the distribution of Comet tails in the histograms in cells exposed to 1 microg/cm2 crocidolite for 6 hr. At higher concentrations of asbestos fibers all the indices in the Comet assay showed significant and irreversible change. All the doses of TNF-alpha caused marginal increase in the mean tail moments. The mean tail moments were highest in the cells with concurrent treatment to TNF-alpha and crocidolite. In the cells pretreated with inhibitors of antioxidant enzymes (aminotriazole for catalase and buthionine sulfoximine for gamma-glutamylcysteine synthetase) asbestos fibers slightly increased oxidant-related fluorescence of dichlorofluorescein (DCFH) but did not cause any further increases in the mean tail moments. This study shows that asbestos fibers cause DNA single strand breaks in human mesothelial cells. Since the inhibition of antioxidant enzymes did not have an effect on the DNA damage caused by the fibers, other mechanisms than free radicals seem to be involved in the induction of DNA damage by mineral fibers.

A multi-institutional study confirms the presence and expression of simian virus 40 in human malignant mesotheliomas.

Exposure to the carcinogen asbestos is a major factor in the development of malignant mesothelioma. However, not all mesotheliomas are associated with asbestos exposure, and only a small minority of people exposed to asbestos develop mesothelioma. Therefore, the identification of the cofactors that render certain individuals more susceptible to asbestos or that cause mesothelioma in people not exposed to asbestos has been a major priority of the International Mesothelioma Interest Group. The possible association of SV40 with mesothelioma was recently discussed in a special session at the Fourth International Mesothelioma Interest Group Conference, and it was decided to conduct a multi-institutional study to independently verify the presence of this tumor virus in mesotheliomas. We report the results of this investigation: (a) DNA and protein analyses revealed SV40 sequences and SV40 large T antigen expression in 10 of 12 mesotheliomas tested (83%); and (b) electron microscopy demonstrated variable amounts of asbestos fibers in 5 (71%) of 7 corresponding lung tissues available for analysis. Our results demonstrate that SV40 DNA is frequently present and expressed in mesotheliomas in the United States. Because our data demonstrate that some patients test positive for both SV40 and asbestos, the possibility that these two carcinogens interact should be investigated in future studies.

Tenascin and fibronectin expression in human mesothelial cells and pleural mesothelioma cell-line cells.

Fibronectin (Fn) and tenascin (Tn) are two major extracellular matrix (ECM) glycoproteins that may have important roles both in fibrotic lung diseases and in lung tumors. The significance of Fn and Tn in human pleural mesothelial cells and pleural diseases is unclear. Transformed human pleural mesothelial cells (Met5A), primary cultures of mesothelial cells, and cultured mesothelioma cell lines were investigated for Fn and Tn immunoreactivity. Mesothelial cells were exposed for 48 to 96 h to transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), amosite asbestos fibers, or oxidants (H2O2 and menadione, a compound that auto-oxidizes to produce superoxide). Immunofluorescence and Western blotting with monoclonal anti-Fn and anti-Tn antibodies, and Northern blotting with a complementary DNA (cDNA) probe for Tn showed that mesothelial cells are capable of producing Fn and Tn. The mRNA level and immunoreactivity of Tn was enhanced by TGF-beta and TNF-alpha, whereas Fn was intensified only by TGF-beta. A wide range of amosite, H2O2, or menadione concentrations had no clear effect on Fn or Tn reactivity. Fn and Tn were present at low or undetectable concentrations in five of six mesothelioma cell lines, whereas the organization of Fn immunoreactivity in these cell lines was variable. Furthermore, results obtained with the tumor tissue of these same mesothelioma patients suggested that Fn and Tn expressions do not necessarily parallel either each other or results obtained with the cultured cells.

DNA single strand breaks and adenine nucleotide depletion as indices of oxidant effects on human lung cells.

The comet assay (single cell gel electrophoresis) is a novel method to assess DNA strand breaks in single cells. We studied the oxidant sensitivity of cultured primary and transformed (MeT-5A) human pleural mesothelial cells, as well as primary and transformed (BEAS 2B) human bronchial epithelial cells, and compared the results obtained with the Comet assay to other markers of oxidant effects on cells, such as depletion of intracellular high-energy nucleotides (ATP, ADP, AMP), accumulation of products of nucleotide catabolism (xanthine, hypoxanthine, uric acid), and release of lactate dehydrogenase (LDH). The cells were exposed for 5 min to 4 h to 50-500 microM H2O2 or to 5-50 microM menadione. Significant tail moment increase, which is a marker of DNA strand breaks in the Comet assay, and intracellular nucleotide depletion occurred simultaneously in MeT-5A and BEAS 2B cells during the first 30-60 min of exposure to H2O2 and menadione. In the Comet assay variation between the individual cells could be detected. LDH release, a marker of cell injury, showed that mesothelial cells were far more sensitive than epithelial cells to oxidant-induced lytic cell injury. MeT-5A and BEAS 2B cells contained similar intracellular antioxidant enzyme activities, which may explain their similar oxidant sensitivity in the Comet assay. A significant increase (164%) in the tail moment was detectable in MeT-5A cells exposed to 50 microM H2O2 for 30 min. This returned to control level during the 4 h of continuing exposure. A 30 min exposure of 25 microM menadione caused a 61% increase in the mean tail moment but, unlike with H2O2, the change was irreversible during the following 4 h incubation. We conclude that human pleural mesothelial cells and bronchial epithelial cells show similar oxidant sensitivity when assessed by the Comet assay, but various oxidants differ in their potency in causing DNA breaks in these cells.

Endogenous antioxidant enzymes and glutathione S-transferase in protection of mesothelioma cells against hydrogen peroxide and epirubicin toxicity.

We have previously shown that cultured malignant mesothelioma cells contain elevated manganese superoxide dismutase (MnSOD) mRNA levels and activities compared with non-malignant mesothelial cells. As many cytotoxic drugs generate both superoxide and hydrogen peroxide, we assessed the relative significance of catalase and the glutathione redox cycle, as well as glutathione S-transferase (GST), in protecting these cells against hydrogen peroxide and epirubicin toxicity. Mesothelioma cell lines containing high (M38K cells) and low (M14K cells) MnSOD, and non-malignant MeT-5A mesothelial cells were selected for the study. M38K cells were the most resistant of these three cell types to hydrogen peroxide (0.1-0.5 mM, 4 h) and epirubicin (0.1-0.5 microg ml(-1), 48 h) as judged by lactate dehydrogenase (LDH) release and by high-energy nucleotide (ATP, ADP, AMP) depletion. Total glutathione was higher in M38K cells (63.8 +/- 20.3 nnmol mg(-1) protein) than in M14K (25.2 +/- 8.2 nmol mg[-1]) or MeT-5A cells (23.5 +/- 4.5 nmol mg[-1]). Furthermore, GST specific activity was higher in M38K cells (111.3 +/- 15.8 U mg[-1]) than in M14K cells (77.4 +/- 6.6 U mg[-1]) or in MeT-5A cells (68.8 +/- 7.6 U mg[-1]). Western blotting indicated the presence of GST-pi in all these cells, the reactivity again being highest in M38K cells. Depletion of glutathione by buthionine sulphoximine and inhibition of catalase by aminotriazole enhanced hydrogen peroxide toxicity in all cell types, while only the depletion of glutathione increased epirubicin toxicity. We conclude that simultaneous induction of multiple antioxidant enzymes can occur in human mesothelioma cells. In addition to the high MnSOD activity, hydrogen peroxide scavenging antioxidant enzymes, glutathione and GST can partly explain the high hydrogen peroxide and epirubicin resistance of these cells in vitro.

Manganese superoxide dismutase in healthy human pleural mesothelium and in malignant pleural mesothelioma.

We hypothesized that manganese superoxide dismutase (MnSOD), known to be induced in rat mesothelial cells by asbestos fibers, cytokines, and hyperoxia, may also be induced in asbestos-related pleural diseases such as mesothelioma. MnSOD was assessed in healthy human pleural mesothelium (n = 6), in biopsy samples of human pleural mesothelioma (n = 7), in transformed nonmalignant human mesothelial cells (Met5A), and in two human mesothelioma cell lines (M14K and M38K) established from the tumor tissue of mesothelioma patients. There was no MnSOD immunoreactivity in five of the six samples of healthy pleural mesothelium, whereas MnSOD immunoreactivity was high in the tumor cells in all the mesothelioma samples. Northern blotting, immunohistochemistry, Western blotting, and specific activity measurements showed lower MnSOD in the nonmalignant Met5A mesothelial cells than in the M14K and M38K mesothelioma cells. In additional experiments the mesothelial and mesothelioma cells were exposed to menadione, which generates superoxide intracellularly, and to epirubicin, a cytotoxic drug commonly used to treat mesothelioma. The M38K mesothelioma cells were most resistant to menadione and epirubicin when assessed by LDH release or by adenine nucleotide (ATP, ADP, and AMP) depletion. These same cells showed not only the highest MnSOD levels, but also the highest mRNA levels and activities of catalase, whereas glutathione peroxidase and glutathione reductase levels did not differ significantly. We conclude that MnSOD expression is low in healthy human pleural mesothelium and high in human malignant mesothelioma. The most resistant mesothelioma cells contained coordinated induction of MnSOD and catalase.

Toxicity and cytogenetic studies of ultrafine titanium dioxide in cultured rat liver epithelial cells.

The in vitro cytotoxicity and the induction of micronuclei of two ultrafine titanium dioxide (TiO(2)) samples was assessed in a rat liver epithelial cell (RLE) assay. Pigmentary TiO(2) was used as a control particle, and mitomycin C, a potent inducer of chromosome damage, was used as a positive control agent in the micronucleus experiments. Since photoexcitation of TiO(2) particles has been reported to increase the cell-killing effect of the dust, a duplicate series of experiments was carried out by irradiating the TiO(2) exposed cells with near-UV light. Neither of the ultrafine TiO(2) samples was toxic to the cells at the concentration range of 5-200 mug/cm(2). The UV treatment had no significant effect on the results. The induction of micronuclei was tested in three concentrations (5, 10 and 20 mug/cm(2)). None of the TiO(2) samples, either ultrafine or pigmentary, increased the numbers of micronuclei in the RLE cells. By contrast, all three samples had a slight decreasing effect on the frequency of micronuclei at the lowest treatment concentration of 5 mug/cm(2), both in the absence and in the presence of UV irradiation. The results suggest that ultrafine particles, similar to pigmentary TiO(2), have no direct clastogenic potential.

Glutathione S-transferase and N-acetyltransferase genotypes and asbestos-associated pulmonary disorders.

BACKGROUND: Humans vary in their ability to metabolize endogenous and exogenous compounds. Glutathione S-transferases (GSTs) and N-acetyltransferases (NATs) are enzymes involved in the detoxification of hazardous agents. The GSTM1 and GSTT1 genes exhibit null (i.e., deletion) polymorphisms; in specific individuals, homozygous deletion (i.e., both copies lost) of these genes can be detected. Polymorphism of the NAT2 gene results in slow and fast acetylators of potentially toxic substances. The GSTM1-null and the NAT2 slow-acetylator genotypes have been associated with increased risks for the development of environmentally induced cancers. PURPOSE: We assessed whether homozygous GSTM1-null or GSTT1-null genotypes or the NAT2 slow-acetylator genotype were associated with increased risks for the development of malignant and nonmalignant asbestos-related pulmonary disorders in a cohort of Finnish construction workers. METHODS: The study population consisted of 145 asbestos insulators who were classified as having been exposed to high levels of asbestos; 69 of these individuals had no pulmonary disorders (control subjects), and 76 had either malignant mesothelioma (n = 24) or nonmalignant pulmonary disorders, such as asbestosis and/or pleural plaques (n = 52). Lymphocyte DNA and the polymerase chain reaction were used to determine the GSTM1, GSTT1, and NAT2 genotypes of the study subjects. Odds ratios (ORs) and 95% confidence intervals (CIs) estimating the relative risks of disease associated with specific genotypes were calculated from 2 x 2 tables by use of Fisher's exact method. RESULTS: Risks for the development of asbestos-related pulmonary disorders were not affected significantly by homozygous deletion of the GSTM1 or GSTT1 genes. However, the risk of developing both malignant and nonmalignant pulmonary disorders for individuals with a NAT2 slow-acetylator genotype was more than twice that observed for those with a NAT2 fast-acetylator genotype (OR = 2.3; 95% CI = 1.1-4.7); the risk of developing malignant mesothelioma for NAT2 slow acetylators was increased almost fourfold (OR = 3.8; 95% CI = 1.2-14.3). Individuals who lacked the GSTM1 gene and possessed a NAT2 slow-acetylator genotype had a risk of developing malignant and nonmalignant pulmonary disorders that was approximately fivefold greater than that observed for those who had the GSTM1 gene and a NAT2 fast-acetylator genotype (OR = 5.1; 95% CI = 1.6-17.6); these individuals had a fourfold increased risk of developing nonmalignant pulmonary disorders (OR = 4.1; 95% CI = 1.1-17.2) and an eightfold increased risk of developing malignant mesothelioma (OR = 7.8; 95% CI = 1.4-78.7) when compared with the same reference group. CONCLUSIONS: Individuals with homozygous deletion of the GSTM1 gene and a NAT2 slow-acetylator genotype who are exposed to high levels of asbestos appear to have enhanced susceptibility to asbestos-related pulmonary disorders.